Bioanalytical methods based on bio-affinity (i.e., the selective binding of two biologically active species, such as antibody/antigen) rely largely on biolabels, labeling techniques and detection of the signals generated from the labels. Biolabels are chemical or biochemical substances that yield, by themselves or through physical/chemical interaction with other reagents, detectable signals that could be correlated to the quantity of the analytes of interest. Biolabels include, but are not limited to, molecules containing radioactive atom(s) (radioactivity), luminescent compounds (emitting light under photoexcitation or by chemical reactions), electroactive compounds (generating electronic signal through redox reactions), magnetic particles (magnetic signal), enzymes (generating detectable species or optical signal via the reaction with substrates). A biolabel includes one or more signal generating unit(s) and one or more reactive group(s). The latter readily form covalent bond(s) with biomolecules to be labeled. Labeling is to link one or multiple labels to a biologically active species to form a complex, which maintain the specific bio-affinity towards the analyte.
Through a cascade of electrochemical redox reactions and follow-up chemical reactions with co-reactants (such as tripropylamine disclosed in U.S. Pat. Nos. 6,451,225 and 6,702,986), electrochemiluminescence (ECL) has became a well-established bioanalytical methodology, in which a metal coordination complex, such as ruthenium (II) polydiimine complex, is used as the signal generating unit in the ECL label molecule. U.S. Pat. Nos. (5,221,605, 5,238,808, 5,310,687, 5,714,089, 5,731,147, 6,140,138, 6,316,607) and a number of research articles disclose ECL labels and their application in immunoassay and DNA probing (e.g., G. F. Blackburn et al., Clin. Chem. 1991, 37/9, 1534-1539; J. H. Kenten et al., Clin. Chem. 1991, 37/9, 1626-1632.).
Improvement of ECL labels has been a long-lasting endeavor. On one hand, there were tremendous attempts to re-design the ruthenium(II) complexes by functionalizing one or more ligand(s) coordinating to the ruthenium(II) ion. On the other hand, efforts in expanding the ECL luminophores from ruthenium(II) complexes to other metal complexes have been extensively made and some are disclosed in patents. For example, U.S. Pat. Nos. 5,858,676 and 6,468,741 disclose the ECL of rare-earth metal complexes and rhenium complexes, respectively. Patent applications WO2012107420, US2013/0323719, US2013/0323857, WO2014019707, WO2014019708 and WO2014019711 disclose new iridium-based complexes as ECL labels.
With respect to the improvement of ruthenium(II) based ECL luminophores, U.S. Pat. No. 5,981,286 discloses ruthenium (II) complexes with hydrophilic bidentate ligands. WO99/15694 discloses a molecular design in which the metal complex luminophore was covalently linked to the co-reactant (i.e., tripropylamine or its analogs). However, because the co-reactant is consumed (rather than recycled) in the process of electrochemiluminescence, its concentration should be much higher than that of the luminophore in a practical electrolyte formulation for bioassays. This approach does not contribute to enhancing ECL intensity. In order to enhance the sensitivity in ECL assays, U.S. Pat. No. 5,679,519 discloses a multi-labeled probe complex comprising a biotinylated bovine serum albumin platform molecule attached by a plurality of electrochemiluminescent labels. It has also been proposed to construct a dendritic scaffold bearing multiple ECL luminophores (US patent application 2005/0059834) and thus achieving multilabeling biomolecules at a single site (see also M. Zhou et al., Anal. Chem. 2003, 75, 6708-6717).
Furthermore, the choice of bidentate ligands has gone beyond the basic 2,2′-bipyridine and 1,10-phenaroline types. U.S. Pat. No. 7,750,157 B2 (EP1759204 A1) discloses a ruthenium(II) label design, in which the bioconjugatable arm is linked to a bidentate ligand that is neither 2,2′-bipyridine nor 1,10-phenaroline.
A common feature of these ruthenium (II) ECL luminophores (without a bioconjugatable group) and ECL labels (with a bioconjugatable group) disclosed in patents or reported in research articles, is that they are composed of a positively charged metal complex luminophore (see FIG. 1A) and a non-luminescent counter anion, such as chloride Cl− or hexafluorophosphate PF6−, etc. The biological activity or the selective binding capability of the species labeled with such lumiophores could be lowered due to the introduction of the positively charged metal complexes. The lowered biological activity enhances non-specific binding in the affinity-based bioassays, and consequently, reduces the sensitivity and the reproducibility of the bioassays. In view of the disadvantage of labels with a positively charged ECL luminophore, U.S. Pat. No. 5,958,783 (CN 1134154, EP 0720614B1) disclosed a type of ECL labels (see FIG. 1B) featuring a charged linker between the positively charged luminophore and the reactive) functional group (i.e., the bioconjugatable group). According to the disclosure, such labels help to improve the performance of ECL immunoassays due to lowered non-specific binding to proteins. But the signal generating unit (the ECL luminophore) in such labels remains a positively charged metal complex, which needs counter ion(s) to neutralize the positive charge.
Towards the same objective of lowering the adverse non-specific binding, U.S. Pat. No. 6,808,939B2 (CN 1612861A) discloses bidentate ligands (i.e., bipyridine and phenanthroline) bearing charged (in particular, negatively charged) functional groups and ruthenium(II) complex labels (see FIG. 1C) consisting of these charged ligands. These bis-heteroleptic labels, having the structure ML′L″2, are negatively charged and have dissociable counter cations in their molecular structure. The results show a significant reduction in non-specific ECL signal when certain negatively charged luminophores were labeled to antibodies. However, while reducing non-specific signal significantly, no improvement of specific signal level was demonstrated.
These ECL luminophores and labels comprising these luminophores are either (predominantly) positively charged or negatively charged with counter ion(s). They are all ionic compounds and can separate or split into cation(s) and anion(s) in electrolyte solutions. Furthermore, these metal complexes that have been synthesized are either bis-heteroleptic or homoleptic metal complexes, i.e., at least two bidentate ligands of the complexes are the same.
L. Della Ciana et al. (J. Phys. Chem. C 2010, 114, 3653-3658) describes a bis-heteroleptic zwitterionic ruthnium(II) luminophore with two identical 2,2′-bipyridine ligands and a bathophenanthroline disulfonate ligand that shows aqueous ECL intensity higher than that of the reference tris(2,2′-bipyridine) ruthenium(II) complex. However, like many other reportedly highly intense ECL luminophores that do not possess a bioconjugatable group, its use in the ECL immunoassay is not feasible, nor is the synthetic approach to introducing a reactive bioconjugatable group to that metal complex established.
The ECL intensity of ruthenium(II) complexes in a specific electrolyte system involving a co-reactant and solvent varies radically with their ligand structures and the combination mode of the ligands. Because of the complexity of the ECL generating process (W. Miao et al, JACS, 2002, 124, 14478-14485), it is also generally accepted that there is no correlation between the ECL intensity and any single property, such as photoluminescence efficiency, redox potential, luminescence wavelength and charging state of the luminophores (M. Zhou et al, Inorg. Chem. 2005, 44, 8317-8325). However, labeling a biological substance with an electronically neutral luminophore does not change the charging state of the biological substance. Thus, among the labels of similar size or similar molecular weight, electronically neutral labels have the least impact on the biological activity of the labeled substance. Therefore, the present invention is concerned with the design of ligand combination modes in order for metal complexes, such as ruthenium(II) complexes, to become an electronically neutral ECL labels that do not dissociate into cation(s) and anion(s) in aqueous buffer solutions or any other solvent.
While we aim to take advantage of the minimal impact a neutral label has on the labeled substance, we have surprisingly found that not only did the neutral ECL labels reduce non-specific signal, some of the neutral ECL labels also generated more intense ECL.
The syntheses of ruthenium(II) complex labels disclosed in prior art followed predominantly an approach (see below) in which a cis-ruthenium diimine (L) dicholoride, i.e., cis-Ru(L)2Cl2, is first synthesized followed by the coordination of a bioconjugatable diimine ligand to the metal ion.RuCl3.xH2O+L→cis-Ru(L)2Cl2   (1)cis-Ru(L)2Cl2+L′→Ru(L)2(L′)Cl2   (2)
U.S. Pat. No. 6,808,939 B2 discloses another synthetic approach (Example 2 thereof), in which a bioconjugatable diimine ligand, i.e., 4-methyl-4′-(3-carboxypropyl)-2,2′-bipyridine, is first coordinated with ruthenium(II) chloride followed by the addition of two identical ligands to form the ECL labels.
These synthetic approaches lead to bis-heteroleptic complex labels which possess two identical non-bioconjugatable ligand and a bioconjugatable ligand. The synthesis of a tris-heteroleptic metal complex ECL label has not been described in prior art.
Unlike the labels with the positively and negatively charged ruthenium(II) luminophores in FIG. 1A-C, the exemplary neutral ECL label in FIG. 1D has three different ligands and could not be synthesized by the method used for the bis-heteroleptic complex labels. Therefore, in addition to ligand combination modes disclosed in this invention, the invention is also directed to the synthetic method by which three different diimine ligands are progressively coordinated with the metal ion to form the tris-heteroleptic ECL labels.
The invention further discloses the biological substances labeled with these neutral ECL luminophores and the use thereof in ECL immunoassay.
It is an object of the present invention to provide luminescent biolabels with electronically neutral metal complex luminophores.
It is another object of the present invention to provide different ligand combination modes that result in the electronic neutrality of metal complex luminophores.
It is a further object of the present invention to provide synthetic approach to the tris-heteroleptic labels with electronically neutral metal complex luminophores.
It is still another object of the present invention to provide substances that are labeled with electronically neutral metal complex luminophores.
It is yet another object of the present invention to provide biolabels to perform quantitative ECL assays of chemical, biochemical and biologically active substances.